Might Hepatitis D Virus Cause More Diseases Than Initially Thought?

Originally published on Forbes on June 16, 2023.

This story is part of a larger series on viroids and virusoids, small infectious RNAs. It is also the seventh installment in a series on hepatitis D virus, a virusoid-like pathogen that causes serious human disease. You may read the others on Forbes or www.williamhaseltine.com

Virusoids are strands of circular infectious RNA. These biological oddities are much smaller than the smallest viruses and have far fewer moving pieces. Most virusoids do not encode proteins, relying entirely on host cellular machinery for replication — what they don’t bring with them, they hijack as needed. Along with depending on the host cell, virusoids also depend on a helper virus to shuttle them between cells and between hosts. This is because virusoids do not come with an envelope, and by extension, any surface proteins of their own. Surface proteins act as a sort of key, allowing viruses to bind to cell membranes and gain access into the interior of the cell. Only once inside can a virus begin replicating.

Hepatitis D virus, despite the name, behaves like a virusoid: it is a small, circular RNA molecule that relies on a helper virus to be shuttled into host cells and relies on host machinery for replication. It is also responsible for the most aggressive form of viral hepatitis, causing severe liver scarring and liver cancer. Hepatitis B virus was long considered its sole helper virus, providing it with the envelope and surface proteins necessary for infection. This also meant the liver was considered the sole site of infection. Novel research challenges both of these assumptions. Published in Nature, the work by Perez-Vargas et al. provides evidence of successful hepatitis D virus transmission via a number of different viral envelopes. The findings indicate hepatitis D virus may be able to persist in a far larger range of tissues than the liver and may contribute to diseases of unknown etiology, including degenerative neurological disease.

Non-Human Hosts: Is Hepatitis B Virus Necessary? 

The origins of hepatitis D virus remain poorly understood. So too the range of species it can infect. Although initially thought to be restricted to human hosts, a growing number of studies report the presence of hepatitis D virus-like RNAs in non-human animals. This includes snakesbirdsfish, insects, and beyond. Granted, the hepatitis D virus-like agents vary significantly from animal to animal, but they retain key characteristics of the pathogen: a small circular genome, a rod-like secondary structure, and hepatitis D antigen proteins (HDAgs).

Crucially, none of the animals infected with the hepatitis D virus-like RNAs were found to carry hepatitis B virus, or any of the other hepadnaviruses. How did they get into the diverse animal cells?

Alternative Viral Envelopes?

The presence of hepatitis D virus-like agents in animals otherwise free of hepadnaviruses raised the possibility that they may have been transmitted with the help of envelopes from other viruses.

To test this, Perez-Vargas et al. exposed liver cells to specially engineered hepatitis D virions; instead of the usual hepatitis B virus envelope and surface proteins, they wrapped hepatitis D virions in the envelope of vesicular stomatitis virus (VSV) or hepatitis C virus (HCV). The envelopes included the surface proteins of each. As a control group, the researchers also created virions with dud envelopes that did not encode any proteins.

Three days after initial infection of the liver cells, hepatitis D virus RNA had accumulated outside of the cells. Usually hepatitis D virus depends on the hepatitis B virus surface proteins for cell exit — essentially, the inverse of the entry process. Yet, the engineered virions with the alternative surface proteins proved just as capable of exiting the cell, as evidenced by the amassment of extracellular RNA. In fact, by day nine the amount of extracellular hepatitis D virus RNA in cell cultures derived from virions coated in the vesicular stomatitis virus envelope was more than sixfold higher than in cell cultures using hepatitis B viral envelopes.

Having determined that hepatitis D virions coated in unconventional envelope proteins could successfully exit the cell, Perez-Vargas et al. next tested to see if the virions could also gain entry into cells and begin replicating. The engineered virions had no trouble, with intracellular levels of hepatitis D virus RNA confirming not only the presence of the virus, but also its successful replication. Indeed, the virions with alternative envelopes could infect kidney cells as well as the usual liver cells. The “normal” hepatitis D virions could not do so.

As is the case with normal hepatitis D virions, the engineered virions entered cells in the same way that their helper viruses did, using the same pathways. Blocking the helper viruses with targeted antibodies also blocked the engineered virions from entry.

The team of researchers then performed the same experiments with an even larger group of helper viruses, creating hepatitis D virions enveloped with the surface proteins of: RD114 cat endogenous virus, murine leukemia virus (MLV), human immunodeficiency virus (HIV), avian influenza virus (AIV), lymphocytic choriomeningitis virus (LCMV), human metapneumovirus (HMPV), dengue virus (DENV), and West Nile virus (WNV). Human metapneumovirus, dengue virus, and West Nile virus were all able to act as helper viruses; levels of hepatitis D virus particle secretion for these alternative envelopes matched those of normal hepatitis D virions. Lymphocytic choriomeningitis virus also managed to act as a helper virus, but the levels of hepatitis D virus particle secretion were lower than when using hepatitis D virions enveloped with hepatitis B virus surface proteins. The remaining viruses failed to enable cell exit and entry.

In Vivo Results

The above experiments were all performed in cell cultures — would the results hold in live animal models?

To find out, mice were co-infected with hepatitis D virus RNA and hepatitis C virus. They were split into three groups: mice first infected with hepatitis C virus and then, four weeks later, with “helper-free” hepatitis D virus; mice first infected with “helper free” hepatitis D virus and then, four weeks later, with hepatitis C virus; and finally, mice co-infected with the two viruses simultaneously.

A few weeks after co-infection, the mice were tested for hepatitis D virus RNA. Hepatitis D virus RNA was detected in all three groups; all mice infected with hepatitis C virus were also positive for hepatitis D virus and significant amounts of hepatitis D RNA was found in their blood. These results suggest that hepatitis C virus can effectively act as a helper virus to hepatitis D virus even in vivo.


Not only can hepatitis D virus be propagated by a variety of helper viruses other than just hepatitis B virus, it can also infect a range of different tissues because of this. This raises the possibility that hepatitis D virus is actually responsible for a far larger number of diseases than initially thought, beyond inflammation of the liver.

© William A. Haseltine, PhD. All Rights Reserved.